Post-translational modifications of the β-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization

2001 ◽  
Vol 354 (2) ◽  
pp. 275-283 ◽  
Author(s):  
Scott M. WARDEN ◽  
Christine RICHARDSON ◽  
John O'DONNELL ◽  
David STAPLETON ◽  
Bruce E. KEMP ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Β Subunit ◽  
Α Subunit ◽  
Amp Activated Protein Kinase ◽  
The Impact

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic α subunit and regulatory β and γ subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the α subunit. The β subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the β-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the β subunit from a particulate extranuclear distribution to a more homogenous cell distribution. Mutation of the serine-108 phosphorylation site to alanine is associated with enzyme inhibition, but no change in cell localization. In contrast, the phosphorylation site mutations, SS24,25AA and S182A, while having no effects on enzyme activity, are associated with nuclear redistribution of the subunit. Taken together, these results indicate that both myristoylation and phosphorylation of the β subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.

scholarly journals Post-translational modifications of the β-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization

2001 ◽  
Vol 354 (2) ◽  
pp. 275 ◽  
Author(s):  
Scott M. WARDEN ◽  
Christine RICHARDSON ◽  
John O’DONNELL Jr ◽  
David STAPLETON ◽  
Bruce E. KEMP ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Cellular Localization ◽  
Post Translational Modifications ◽  
Amp Activated Protein Kinase

scholarly journals Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

2005 ◽  
Vol 386 (2) ◽  
pp. 237-244 ◽  
Author(s):  
Hamid R. SAMARI ◽  
Michael T. N. MØLLER ◽  
Lise HOLDEN ◽  
Tonje ASMYHR ◽  
Per O. SEGLEN
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Calyculin A ◽  
Β Subunit ◽  
Isolated Rat Hepatocytes ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

scholarly journals Diverse Cytopathologies in Mitochondrial Disease Are Caused by AMP-activated Protein Kinase Signaling

2007 ◽  
Vol 18 (5) ◽  
pp. 1874-1886 ◽  
Author(s):  
Paul B. Bokko ◽  
Lisa Francione ◽  
Esther Bandala-Sanchez ◽  
Afsar U. Ahmed ◽  
Sarah J. Annesley ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitochondrial Disease ◽  
Mammalian Cells ◽  
Energy Homeostasis ◽  
Mitochondrial Diseases ◽  
Dependent Manner ◽  
Α Subunit ◽  
Gene Dose ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK α subunit (AMPKαT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKα expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPKα expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial “mass” and ATP levels were reduced by AMPKα antisense inhibition and increased by AMPKαT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKα antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.

scholarly journals Kinase-independent transcriptional co-activation of peroxisome proliferator-activated receptor α by AMP-activated protein kinase

2004 ◽  
Vol 384 (2) ◽  
pp. 295-305 ◽  
Author(s):  
Myriam BRONNER ◽  
Rachel HERTZ ◽  
Jacob BAR-TANA
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Phosphorylation Site ◽  
Atp Depletion ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Α Subunit ◽  
Co Activation ◽  
Amp Activated Protein Kinase

AMPK (AMP-activated protein kinase) responds to intracellular ATP depletion, while PPARα (peroxisome proliferator-activated receptor α) induces the expression of genes coding for enzymes and proteins involved in increasing cellular ATP yields. PPARα-mediated transcription is shown here to be co-activated by the α subunit of AMPK, as well as by kinase-deficient (Thr172Ala) and kinase-less (Asp157Ala, Asp139Ala) mutants of AMPKα. The Ser452Ala mutant of mPPARα mutated in its putative consensus AMPKα phosphorylation site is similarly co-activated by AMPKα. AMPKα or its kinase-less mutants bind to PPARα; binding is increased by MgATP, to a lesser extent by MgADP, but not at all by AMP or ZMP [AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) monophosphate]. ATP-activated binding of AMPKα to PPARα is mediated primarily by the C-terminal regulatory domain of AMPKα. PPARα co-activation by AMPKα may, however, require its secondary interaction with the N-terminal catalytic domain of AMPKα, independently of its kinase activity. While AMPK catalytic activity is activated by AICAR, PPARα co-activation and PPARα-controlled transcription are robustly inhibited by AICAR, with concomitant translocation of nuclear AMPKα or its kinase-less mutants to the cytosol. In conclusion, AMPKα, independently of its kinase activity, co-activates PPARα both in primary rat hepatocytes and in PPARα-transfected cells. The kinase and transcriptional co-activation modes of AMPKα are both regulated by the cellular ATP/AMP ratio. Co-activation of PPARα by AMPKα may transcriptionally complement AMPK in maintaining cellular ATP status.

scholarly journals Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade

2007 ◽  
Vol 403 (1) ◽  
pp. 139-148 ◽  
Author(s):  
Matthew J. Sanders ◽  
Pascal O. Grondin ◽  
Bronwyn D. Hegarty ◽  
Michael A. Snowden ◽  
David Carling
Keyword(s):  
Protein Kinase ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Inhibitory Effect ◽  
Allosteric Activation ◽  
Α Subunit ◽  
Γ Subunit ◽  
New Findings ◽  
Amp Activated Protein Kinase ◽  
Dependent Pathway

AMPK (AMP-activated protein kinase) is activated allosterically by AMP and by phosphorylation of Thr172 within the catalytic α subunit. Here we show that mutations in the regulatory γ subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of Thr172 by PP (protein phosphatase)2Cα. Moreover, a mutation in the γ subunit almost completely abolishes the inhibitory effect of AMP on dephosphorylation. We were unable to detect any effect of AMP on Thr172 phosphorylation by either LKB1 or CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver, there was an apparent AMP-stimulation of Thr172 phosphorylation by LKB1, but this was blocked by the addition of NaF, a PP inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2Cα in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of Thr172 were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2Cα, present in the preparations of the kinases used in the earlier studies. Taken together, our results demonstrate that AMP activates AMPK via two mechanisms: by direct allosteric activation and by protecting Thr172 from dephosphorylation. On the basis of our new findings, we propose a simple model for the regulation of AMPK in mammalian cells by LKB1 and CaMKKβ. This model accounts for activation of AMPK by two distinct signals: a Ca2+-dependent pathway, mediated by CaMKKβ and an AMP-dependent pathway, mediated by LKB1.

scholarly journals The regulation of AMP-activated protein kinase by phosphorylation

2000 ◽  
Vol 345 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Silvie C. STEIN ◽  
Angela WOODS ◽  
Neil A. JONES ◽  
Matthew D. DAVISON ◽  
David CARLING
Keyword(s):  
Protein Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Aspartic Acid Residue ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Mutant Complex

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr172) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr172 on AMPK activity. Mutation of Thr172 to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr172 to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the α subunit and one site on the β subunit. Furthermore, we provide evidence that phosphorylation of Thr172 may be involved in the sensitivity of the AMPK complex to AMP.

scholarly journals Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

2013 ◽  
Vol 288 (23) ◽  
pp. 16495-16505 ◽  
Author(s):  
Huaiping Zhu ◽  
Cate M. Moriasi ◽  
Miao Zhang ◽  
Yu Zhao ◽  
Ming-Hui Zou
Keyword(s):  
Protein Kinase ◽  
Splice Variants ◽  
Short Form ◽  
Phosphorylation Site ◽  
Nucleocytoplasmic Transport ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Ampk Activation ◽  
Downstream Target ◽  
Amp Activated Protein Kinase

Two splice variants of LKB1 exist: LKB1 long form (LKB1L) and LKB1 short form (LKB1S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1L) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1S. Substitution of Ser-399 to alanine did not alter the activity of LKB1S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1L), Ser-399 (in LKB1S) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1S and consequent AMPK activation.

scholarly journals LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

2000 ◽  
Vol 345 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Sean P. COLLINS ◽  
Junewai L. REOMA ◽  
David M. GAMM ◽  
Michael D. UHLER
Keyword(s):  
Protein Kinase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Multiple Cancer ◽  
Dependent Protein Kinase ◽  
Rnase Protection ◽  
Camp Dependent Protein Kinase ◽  
Increased Risk ◽  
Dependent Protein

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

scholarly journals Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts

2012 ◽  
Vol 49 (1) ◽  
pp. 47-55 ◽  
Author(s):  
H Tokuda ◽  
K Kato ◽  
H Natsume ◽  
A Kondo ◽  
G Kuroyanagi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Interleukin 6 ◽  
P38 Map Kinase ◽  
Thrombin Time ◽  
Rt Pcr ◽  
Α Subunit ◽  
Compound C ◽  
Kinase C ◽  
Amp Activated Protein Kinase

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression ofIL6mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. TheIL6mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

scholarly journals Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

2018 ◽  
Vol 293 (44) ◽  
pp. 16994-17007 ◽  
Author(s):  
Xin Gu ◽  
Michael D. Bridges ◽  
Yan Yan ◽  
Parker W. de Waal ◽  
X. Edward Zhou ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Energy Homeostasis ◽  
Metabolic Diseases ◽  
Kinase Domain ◽  
Carbohydrate Binding ◽  
Conformational Heterogeneity ◽  
Α Subunit ◽  
Distance Distributions ◽  
Double Electron Electron Resonance ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the β-subunit. Here, using double electron–electron resonance (DEER) spectroscopy, we demonstrate that the CBM–KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD–CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD–CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.

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